Liquid-liquid extraction in aqueous systems has received much attention for separation or purification of biomaterials and other products. In this specification, "biomaterial" means any water soluble or water insoluble, bio-affecting substance, whether of biological or non-biological origin. Biomaterials include compounds, molecules, polymers, particles and simple as well as complex substances which exist in nature or which may be synthesized by biological or non-biological processes. The term thus includes proteinaceous substances (such as albumins and enzymes), amino acids, nucleic acids, peptides and polypeptides, antibodies, antigens, steroids, hormones, cells, cell fragments, extracts and debris, fermentation products and derivatives, and pharmaceuticals of all kinds, including antibiotics, polymeric carbohydrates, and the like.
Although the separation and purification of useful biomaterials is a significant concern of the present invention, the invention is also applicable to monbiomaterials, such as pigments, particulate matter and organic solutes of all kinds. Accordingly, as used hereinafter, the term "product(s)" means useful biomaterials or non-biomaterials which can be separated by the methods of the invention.
Liquid-liquid extraction with at least two aqueous phases is especially appropriate for separation and purification of biomaterials from aqueous systems because aqueous phase systems, as compared with immiscible organic-water phase systems, are mild in their response to biomaterials, primarily due to high water content and the ability to sustain physiological conditions in the systems due to the presence of sugars, salts, buffers and other low molecular weight substances. Accordingly, aqueous liquid-liquid extraction systems are effective for partitioning of complex and fragile substances, including cells and cell constituents, while retaining requisite viability and/or biological activity.
In general terms, aqueous liquid-liquid extraction involves addition to water of two or more substances (extractants) which are water-soluble but mutually immiscible. To this system is then added a mixture containing the material which it is desired to extract. The aqueous mixture comprising the extractants and the material to be extracted is then agitated and permitted to equilibrate, whereupon the extractants separate into liquid layers accompanied by partitioning of the desired material into one or several of the phases or interfaces thereof. If the material to be extracted is water soluble, it will partition primarily into one or more of the liquid phases. Water insoluble or particulate material, such as cells or cell fragments, may partition not only to one or more of the liquid phases but also to any interface between the phases.
Taking a two liquid aqueous phase system as representative, the distribution of a material to be extracted is defined by the partition coefficient, K.sub.p, as the ratio of equilibrium concentration of the partitioned material in the top and bottom phases, respectively: K.sub.p =C.sub.t /C.sub.b (Equation 1). Since partitioning depends upon the properties not only of the extractants constituting the liquid phases but also on characteristics of the partitioned solute, especially surface properties, the partition coefficient K.sub.p is the sum of several contributing factors: EQU 1n K.sub.p =1n k.sub.el.sup.+1n k.sub.hydrophob.sup.+1n k .sub.hydrophil.sup.+1n EQU k.sub.conf.sup.+k.sub.lig (Equation 2)
where k.sub.el,k.sub.hydrophob, k.sub.hydrophil, k.sub.conf and k.sub.lig are the partition coefficient factors due to electrical, hydrophobic, hydrophilic, conformational and ligand effects, respectively. Albertsson, J. of Chromatography 159:111-122(178); M. Ramstorp, "Novelty Affinity Techniques," Thesis, Department of Pure and Applied Biochemistry, University of Lund, Sweden, 1983, Page 26.
If the partitioned material is particulate, other factors will contribute to the partition coefficient because of the possibility of partitioning of the particulate matter to the interface between the liquid phases (Albertsson, Advance in Protein Chemistry 24:309-341, 1970). It will be evident from the composite partition coefficient (Equation 2) that partitioning can be substantially affected by changes in hydrophobic and hydrophilic character as well as by charge and material density, structure, molecular weight, molecular weight distribution, the presence of ligands for the solute, and biospecific attraction.
Modification of partitioning behavior for the purpose of enhancing the proportion of material which concentrates in a given phase of a liquid-liquid extraction system is therefore a highly desirable objective, particularly if it can be achieved, in the case of biomaterials, without substantial injury to the partitioned material, either by reason of loss of compositional or structural integrity or because of diminution of biological activity.
Heretofore, desired improvements in liquid-liquid extraction have focused on selection of the extractants, as in U.S. Pat. No. 4,144,130 to Kula et al and 4,508,825 to Kim et al. In the Kula et al patent, as further explained in H. Hustedt et al, Biotechnology and Bioengineering, Volume XX, 1989-2005 (1978), enzymes are effectively recovered from cells and cell fragments by multiphase liquid separation wherein the extractant liquids comprise the combination of a high molecular weight compound and an inorganic salt, or the combination of at least two high molecular weight compounds. Representative of the first combination is a polyethylene glycol (PEG)-potassium phosphate buffer (PPB) pair or a PEG-dextran pair.
In a similar approach, Kim et al separate extracellular protease and amylase from a fermentation broth by the addition of PEG and a cationic epihalohydrin-polyamine copolymer or a dextran polymer.
In another approach, focusing on purification of specific proteins, Johansson modified the PEG by attachment of functional groups which introduced surface charges. He was thus able to improve the partitioning of three different albumins. Biochim. Biophys. Acta, 222 (1970), 381,389.
Jizomoto improved the separation of animal tissue albumin and gum arabic by varying the molecular weight of the polyethylene glycol extractant. J. Pharmaceutical Sci. 74, No. 4, Apr. 1985, 469-472.
Others have employed affinity partitioning for purification of proteins and other substances by adding a material capable of coupling to the polymeric extractant, the resulting polymer-ligand then partitioning predominately into one of the liquid phases. Flanagan et al, J. of Biological Chemistry, 250, No. 4, Feb. 25, 1975, 1484-1489.
Still others have examined the effect of pH on a system and have determined that proteins can be forced to partition according to their isoelectric points as a result of adding charged polymers. Johansson et al, European Journal Biochemistry, 33,379 (1973).
Nevertheless, despite the advances in the art, the known extraction systems are limited in their applicability, and only specific partitionable materials (such as certain proteins) have benefitted. Accordingly, a method of enhancing liquid-liquid extraction having more general applicability and capability of large scale operation will have substantial value.